Mycoattractants and mycopesticides

ABSTRACT

The present invention utilizes extracts of the pre-sporulation (preconidial) mycelial stage of entomopathogenic fungi as insect attractants and/or pathogens. The fungus can be cultivated on grain, wood, agricultural wastes or other cellulosic material. More than one fungus and substrate can be used in combination.

This application is a divisional of U.S. patent application Ser. No.09/969,456, filed Oct. 1, 2001, which is a continuation-in-part of U.S.patent application Ser. No. 09/678,141, filed Oct. 4, 2000, now issuedas U.S. Pat. No. 6,660,290.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to mycology, entomology and the use offungal mycelium as insect attractants (mycoattractants) andbiopesticides (mycopesticides). More particularly, the invention relatesto the control and destruction of insects, including termites, fireants, carpenter ants, flies, beetles, cockroaches, grasshoppers andother pests, using pre-sporulation fungal mycelium as an attractantand/or infectious agent.

2. Description of the Related Art

Insects are among the most diverse and numerous life forms on earth.While the majority of the one million named species of insects areconsidered beneficial, somewhere from 1% to 5% are considered to bepests. These insect pests cause tremendous losses in terms of directdestruction of crops, livestock and human dwellings and vector pathogensincluding protozoans, bacteria, viruses and rickettsia that causedevastating human health problems. The physical, mental, economic,social, and ecological implications of these pest insects areimmeasurable on any scale.

The use of chemical pesticides is the cause of many secondaryenvironmental problems aside from the death of the targeted pest.Numerous problems attributed to chemical pesticides are caused orcompounded by widespread application necessitated by lack of suitablemeans of attracting the targeted pest to the pesticide. Communities areincreasingly in need of natural solutions to pest problems.

Compounding these problems, many pest type or vermin insects havedeveloped a broad spectrum of resistance to chemical pesticides,resulting in few commercially available pesticides that are effectivewithout thorough and repeated applications. In addition to being largelyineffective and difficult and costly to apply as presently utilized,chemical pesticides present the further disadvantage of detrimentaleffects on non-target species, resulting in secondary pest outbreaks.Widespread use of broad-spectrum insecticides may destroy or greatlyhamper the natural enemies of pest species, with pest speciesreinfesting the area faster than non-target species, thereby allowingand encouraging further pest outbreaks. Van Driesche, R. G. and T. S.Bellows Jr., Biological Control, Chapman & Hall, pp. 4-6 (1996). Furtherexacerbating these problems, introduced “alien” insect pests such astermites or fire ants often have few or no natural enemies. There is aparticular need for natural alternatives.

Biological control agents have been tried with varying results. Bacteriasuch as Bacillus thuringiensis are used with some success as a spray onplants susceptible to infestation with certain insects. Fungal controlagents are another promising group of insect pathogens suitable for useas biopesticides. However, limited availability, lack of effectivedelivery systems, reliability and cost has hampered the development ofsuch fungal control agents. Host range and specificity has been aproblem as well as an advantage: a fungal pathogen that is pathogenic(capable of causing disease) and virulent (in the sense of beingextremely infectious, malignant or poisonous) to one insect species maybe ineffective against other species, even closely related species ofthe same family or genus. However, some success has been demonstrated.

The typical lifecycle of the entomopathogenic (capable of causing insectdisease) fungi is thought to involve adhesion of the spore(s) to thehost insect cuticle, spore germination, penetration of the cuticle priorto growth in the hemocoel, death, saprophytic feeding, hyphalreemergence and sporulation. For example, U.S. Pat. No. 6,254,864 (2001)to Stimac et al. discloses dry powder Beauveria bassiana spore andspore/mycelium compositions for control of cockroaches and antsincluding carpenter ants, pharaoh ants and fire ants. U.S. Pat. No.4,925,663 (1990) to Stimac discloses Beauveria bassiana used to controlfire ants (Solenopsis). Rice, mycelia and spores (conidia) mixture maybe applied to fire ants or used as a bait and carried down into thenest, thereby introducing spores. U.S. Pat. No. 5,683,689 (1997) toStimac et al. discloses conidial control of cockroaches, carpenter ants,and pharaoh ants using strains of Beauveria bassiana grown on rice. U.S.Pat. No. 5,413,784 (1995) to Wright et al. discloses compositions andprocesses directed to the use of Beauveria bassiana and Paecilomycesfumosoroseus to control boll weevils, sweet potato whiteflies and cottonfleahoppers. U.S. Pat. No. 5,728,573 (1998) to Sugiura et al. disclosesgerminated fungi and rested spore termiticides of entomogenous fungussuch as Beauveria brongniartii, Beauveria bassiana, Beauveria amorpha,Metarhizium anisopliae and Verticillium lecanii for use against insectssuch as termites, cockroaches, ants, pill wood lice, sow bugs, largecentipedes, and shield centipedes. U.S. Pat. Nos. 5,939,065 (1999) and6,261,553 (2001) to Bradley et al. discloses conidial formulations ofBeauveria and methods for control of insects in the grasshopper family.U.S. Pat. No. 4,942,030 (1990) to Osborne discloses control ofwhiteflies and other pests with Paecilomyces fumosoroseus Apopka sporeconidia formulations. The Paecilomyces fungus is also useful for controlof Diptera, Hymenoptera, Lepidoptera, Bemisia, Dialeurodes, Thrips,Spodoptera (beet army worm), Leptinotarsa (Colorado potato beetle),Lymantria (Gypsy moth), Tetranychus, Frankliniella, Echinothrips,Planococcus (citrus mealybug) and Phenaococcus (solanum mealybug). U.S.Pat. No. 5,360,607 to Eyal et al. discloses prilled Paecilomycesfumosoroseus compositions utilizing mycelium grown via submergedfermentation to produce conidia to control various insects includingwhiteflies, mosquitoes, aphids, planthoppers, spittlebugs, mites,scales, thrips, beetles or caterpillars. U.S. Pat. No. 5,165,929 (1992)to Howell discloses use of Rhizopus nigricans and other fungus in theorder Mucorales as a fungal ant killer. U.S. Pat. No. 5,989,898 (1999)to Jin et al. is directed to packaged fungal conidia, particularlyMetarhizium and Beauveria. The scientific journal literature alsodiscusses similar uses of conidial preparations.

One disadvantage to such approaches is that the fungal lifecycle may beparticularly sensitive to and dependent upon conditions of humidity,moisture and free water, particularly during the stages of sporegermination and sporulation after death of the insect.

A particular disadvantage with conidial preparations becomes apparentfrom U.S. Pat. No. 5,595,746 (1997) to Milner et al. for termitecontrol, which discloses Metarhizium anisopliae conidia utilized as atermite repellant in uninfested areas and as a termite control method ininfested areas. The difficulties of utilizing conidia orconidia/mycelium as a bait and/or contact insecticide are readilyapparent when considering that conidia are effective as an insectrepellant to termites and are repellant in varying degrees to most orall targeted insect pests. A repellant, of course, does not facilitateuse as a bait or contact insecticide. This may be a factor in explainingwhy fungal insecticides have all too often proven more effective in thelaboratory, where conidia may be unavoidable in the testing chamber oreven directly applied to insects, than in the field.

U.S. Pat. No. 5,888,989 (1999) to Kern discloses synergisticcombinations of conidia of entomopathogenic fungi such as Beauveria andMetarhizium with parapyrethroid insect compositions such as silafluofenand etofenprox, nitromethylenes such as imidacloprid, carbamates such asfenoxycarb and phenylpyrazoles such as fipronil. Problems remain withthe repellency of the spores, the repellency of the pesticides and theuse of conidia as a vector of infection.

Certain sexually reproducing brown-rot fungi (such as Lenzites trabea),dry rot fungi and other fungi are known to influence termite behaviorsin laboratory and field tests, demonstrating attractant properties,eliciting trail-following, etc. See, for example, U.S. Pat. No.4,363,798 (1982) to D'Orazio for termite baits utilizing brown rotfungus as an attractant mixed with toxicant boron compounds. Thebrown-rot fungi Lentinus lepideus and aqueous extracts of this fungiwere found to be extremely lethal to termites in the laboratory, U.S.Pat. No. 3,249,492 (1966) to Lund. Certain fungi are known to producesubstances that elicit trail-following in Rhinotermitidae in thelaboratory, i.e., Gloephyllum trabeum, Oligoporous balsameus and Serpulalacrimans. Various extracts of the sexually reproducing Zygomycetesfungus Micromucor ramannianus and other fungi coexisting withReticulitermes have also been shown to exhibit phagostimulatory (feedingstimulatory) effects and phagodeterrent effects. See U.S. Pat. No.6,203,811 (2001) to McPherson et al. However, there remains a need forimproved fungal attractants and pesticides.

The fresh, dried and rehydrated mycelium of entomopathogenic fungi hasbeen utilized as a spore source in both the laboratory and field. See,for example, the U.S. patents above, where conidia are directly orindirectly produced from solid substrate or liquid fermentor grownmycelium. Pre-sporulation mycelium of Metarhizium anisopliae,Metarhizium flaviride, Beauveria bassiana, Paecilomyces farinosus,Paecilomyces lilacinus and Hirsutella citriformis has also been utilizedas a spore source in agricultural fields for use against varioussubterranean and agricultural pests including the black vine weevil, thecranberry girdler (Chrysoteuchia topiaria), sod webworm, rice brownplanthopper, stem borer, European corn borer and fall armyworm. SeeBooth and Shanks Jr., Potential of a Dried Rice/Mycelium Formulation ofEntomopathogenic Fungi to Suppress Subterranean Pests in Small Fruits,Biocontrol Science and Technology, 8: pp. 197-206 (1998); Rombach etal., Infection of Rice Brown Planthopper, Nilaparvata lugens (Homoptera:Delphacidae), by Field Application of Entomopathogenic Hyphomycetes(Deuteromycotina), Environmental Entomology, 15(5): pp. 999-1110 (1986);and Maniania, Evaluation of three formulations of Beauveria bassiana(Bals.) Vuill. for control of the stem borer Chilo partellus (Swinhoe)(Lep., Pyralidae), Journal of Applied Entomology, 115: pp. 266-272(1993). Pre-sporulation vegetative mycelium has also been a focus withthe Entomophthorales mycopesticidal fungi such as Zoophthora radicans,which produce fragile, thin-walled spores that are difficult to massproduce, harvest and formulate on an industrial scale, thus leading toinvestigations of the also somewhat delicate and ephemeral mycelium.After being applied to the crop or soil, the mycelium produces sporesthat infect the target pests. See, for example, U.S. Pat. No. 4,530,834(1985) to McCabe et al. Pre-sporulation mycelium of Hirsutellacitriformis has been also been utilized in the field as conidia of thefungi are difficult to produce due to low sporulation rates, slimeproduction of the mycelium and irregular growth patterns.

Another continuing problem with existing techniques for combating pestsincluding social insects has been inconsistent bait acceptance. Baitsare often bypassed and left uneaten by social insects such as termitesand carpenter ants, which are hard to attract. Such may be a particularproblem with insects such as termites and carpenter ants, as opposed tohouse ants and cockroaches, because it is usually not possible to removecompeting food sources for termites and carpenter ants. Attractants,pheromones and feeding stimulants have sometimes increased theconsistency of bait acceptance, but such increases cost and complexity,and there remains a continuing need for improved baits with improvedbait acceptance.

There is, therefore, a continuing need for improved attractants andbaits in general. There is a continuing need for enhancing theeffectiveness of entomopathogenic fungal biopesticide products andmethods and enhancing the attractiveness of such fungal pesticides toinsects. There is also a need for improved packaging, shipping anddelivery methods.

In view of the foregoing disadvantages inherent in the known types ofinsect control agents, the present invention provides improved fungalbiocontrol agents and methods of using such agents.

SUMMARY OF THE INVENTION

The present invention offers an environmentally benign approach toinsect control by attracting insects that contact or ingest“preconidial” mycelium of mycopesticidal/entomopathogenic fungi (thatis, mycelium in a developmental state prior to conidia or sporeformation). Such preconidial mycelium may be used solely as anattractant (either as an attractant for pest insects or as an attractantfor beneficial insects) or as an attractant and pathogen where thepreconidial mycelium is both the attractant and the pathogenic agent.Where attractant mycopesticidal strains are utilized with socialinsects, the infected insects carrying the fungal hyphae become a vectorback to the central colony, further dispersing the mycopesticidalmycelium. The preconidial mycopesticidal mycelium can grow within aninsect, can grow via spread to another insect or can grow via spread toan organic debris housing and subsequent insect infestation. Thus,multiple avenues of growth and infection are provided while entirelyavoiding the supposed “necessity” of conidia germination as a means ofinfection.

The preconidial mycelium of mycopesticidal fungi is grown in pureculture using standard techniques for in vitro propagation. Onceinoculated onto a substrate such as grain or wood, the mycelia maturesto a state prior to conidia formation. The window of utility extendsfrom post-spore germination through all stages of mycelial growth priorto sporulation. The preconidial mycelium may be utilized as is or may bearrested in its development through means such as flash chilling,freeze-drying, air-drying, refrigeration or gaseous cooling and packagedin spoilage-proof or sealed packages. The end-user facilitates openingthe package and placing the exposed mycelia contents in the vicinity ofrecent pest activity. For use as an attractant, extracts of thepreconidial mycelium may also be utilized. It is envisioned that thefungal attractants and/or pesticides may be used in conjunction with anytype of appropriate trap or attractant disseminator or delivery systemas is known to the art.

The present invention thus provides improved products and methodswherein the fungal mycelium acts as food and attractant and/or as aningested or contact insecticide, palatable enough that insects willreadily consume it even in the presence of competing food sources, withhigh recruitment of other insects among insects that exhibit suchbehavior. This results in multiple visits to a highly attractive (andpotentially virulent) food, thereby providing numerous individual insectand/or colony vectors of inoculation.

The present invention further provides these and other advantages withimproved control of insect pests using fungal compositions(mycopesticides and mycoattractants) having strong attractant propertiesand placing these attractant preconidial fungi in or around an object orarea to be protected. The present invention also provides insecticidalfoods and baits which utilize, as a toxicant, relatively innocuous andnaturally occurring materials as the active agent, so as to controlinsects without undue effect on the ecology. Alternatively, the presentinvention provides attractants which can be utilized with biocontrolagents, environmentally benign biopesticides, chemical control agentsincluding insect toxicants and pesticides, physical control agents suchas mechanical and electrical devices and combinations thereof.

A further advantage is achieved by actively avoiding the use of conidiain that the time and expense of raising conidial stage mycelium and/orseparating conidia is rendered unnecessary and avoided.

Still further objects and advantages of the present invention willbecome more apparent from the following detailed description andappended claims.

Before explaining the disclosed embodiments of the present invention indetail, it is to be understood that the invention is not limited in itsapplication to the details of the particular products and methodsillustrated, since the invention is capable of other embodiments,including those embodiments which have not yet been reduced to practiceand tested. In addition, the terminology used herein is for the purposeof description and not of limitation.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Fungi have been recognized as the causative agents of insect diseasesand the fungal spores utilized as microbial insecticides for over 100years. A great deal of ongoing research continues to be directed to theuse of spores as mycopesticides; see, for example, U.S. Pat. No.6,261,553 (2001) to Bradley et al. and U.S. Pat. No. 6,254,864 (2001) toStimac et al. Attention has continued to be directed to spores as theinfectious agent, perhaps because the prevailing paradigm has been thatthat infection is generally via spores (infective propagules that aretermed spores or conidia in Zygomycotina and Deuteromycotina,microconidia in certain entomophthoralean species such as Conidioboluscornatus, zoospores in Mastigomycotina, or plantons or acospores inAscomycotina, including resting spores) with subsequent sporegermination and hyphal penetration of the host body by the fungalmycelium causing insect death. See, for example, U.S. Pat. Nos.4,925,663, 5,360,607, 5,413,784, 5,683,689 and 6,254,864 andSchmid-Hempel, P., Parasites in Social Insects, Princeton UniversityPress, pp. 36, 43-44 (1998). The fungal mycelium itself, particularlythat of the Deuteromycetes, has been utilized only as a spore source,whether in the laboratory or in the field.

In contrast to the previous research, the present inventor has foundthat prior to spore or conidia formation, the preconidial mycelium ofentomopathogenic, insect-killing fungal species possesses numerouspreviously unrecognized properties as an attractant and as a uniquelyenticing insect food composition, capable of inducing novel behaviors inthe social insects including “grazing” on and “housekeeping” inpreconidial mycelium and scattering of the preconidial mycelium aroundfeeding areas and nesting chambers. The preconidial mycelium of virulentstrains can act as an infectious agent with numerous vectors ofinfection and infestation via ingestion and/or contact with furthermycelial growth.

The concepts of “pathogens” and “pathogenic” (and the related“entomopathogens” and “entomopathogenic”) have implications that extendwell beyond the standard dictionary definition of “capable of causingdisease or mortality.” Some entomopathogenic fungi are widespread andcause no known effects whatsoever in their insect hosts;Myrmicinosporidium durum is illustrative of entomopathogenic fungi thatcause few symptoms and are consequently hard to detect in the firstplace. Schmid-Hempel, supra, p. 83 (1998). Entomopathogenic fungi asused herein are those capable of infecting and parasitizing insects,regardless of their actual effect on the host. “Virulence” and “virulentstrains” similarly have meanings extending beyond the dictionarydefinition of extremely infectious, malignant or poisonous. Parasitevirulence and host resistance determine how host and parasite interactin ecological time and how they both coevolve. Virulence is oftendefined as an increase in the host mortality rate as a result of theparasite's presence. But reduced host fecundity, parasite replicationrate within the host, and several other measures have also been used.Virulence should in principle also include instances where the behaviorof the host is manipulated by the parasite to increase the probabilityof its successful transmission and where it places the individual hostat greater risk. See Schmid-Hempel, supra, pp. 237-238. Here the termsvirulent and virulence are used in a broad sense that encompasses all ofthese meanings. It will refer to processes that are caused byentomopathogenic fungi and which lead to a reduction in some componentof the host's fitness or mortality. Virulence and resistance aretherefore properties that emerge as a result of host-parasiteinteraction in a given environment. Expression of virulence is asdiverse as the lifestyles and characteristics of the insect hosts andthe entomopathogenic fungi themselves. The present invention providesimproved mycoattractants and mycopesticides (fungal mycelia utilized asinsect attractants or baits and/or insect biopesticides, after mycology,the study of fungi). The attractiveness of fungal mycelia to manyspecies is well known. Black Angus cows have been observed runninguphill (a rare event) to reach spent Oyster mushroom mycelium on straw.Cultured mycelia such as Morel mycelium is considered a delicacy whenadded to human foods; gourmet mushrooms themselves are a structurearising from mycelium to form fruitbodies. Indeed, the attractiveness ofmycelial scents is to a great degree responsible for the fresh andrefreshing scent of a forest after a rain, a result of the mushroommycelia responding to the humid conditions with rapid growth. Myceliumis also known to be highly attractive to insects. Certain leaf-cuttingants, termites and wood-boring beetles are known to cultivate and raisefungal mycelium as an exclusive food source (for example, “ambrosiafungi”) and mycelium is a preferred food source of many insect species.As discussed above, brown rot mycelium (the mycelial stage of awood-rotting type of fungus that produces some mushrooms) has been usedas an attractant for termites.

However, for use as a “contact insecticide” control agent, applicationof the fungal entomopathogenic species has typically involved eitherconidia (spores) or a mixture of conidia and mycelium or mycelium as aspore source in the laboratory or field. Such conidial contactinsecticides suffer from at least two major biological disadvantages: 1)conidia and conidia/mycelium preparations are to some degreeunattractive or even repellant to insects; and 2) such conidiapreparations are highly dependent on free water or humid conditionsand/or specific insect recognition factors for gestation of the sporesand infestation during the typical life cycle of an insect fungalcontrol agent. Furthermore, conidia have been found to be more effectiveagainst “stressed” insects and/or insect populations than againsthealthy insects and populations. Laboratory procedures for testingentomopathogenic fungi often involve procedures inapplicable in thefield, such as “dusting” of many or all of the insects with spores orforced contact with conidia in petri dishes (itself a form a stress).Insects infected with mycopesticidal spores are often rejected orisolated from the general population, thus limiting the furtherspreading of the fungal disease. Wilson, E. O., The Insect Societies,The Belknap Press of Harvard University Press, pp. 103-119 (1974). Forthese and other reasons, conidia of entomopathogenic fungi have oftenbeen much more effective under laboratory conditions than in the field.

Noting that conidia have been utilized as a repellant for termites, anddriven by a desire to avoid contamination of a sterile-culture gourmetand medicinal mushroom laboratory with the spores of mycopesticidalspecies, further investigation of the preconidial stages of theDeuteromycetes Metarhizium and Beauveria were undertaken. Thepreconidial stage is the vegetative stage of the fungus, prior to theformation of structures leading to the release of airborne spores (whichis distinguished from fragmentation of hyphae which can become airborneif dried). Those skilled in the art will recognize that mycelia ormycelial hyphal fragments may form structures such as arthrospores (apreconidial structure imbedded within the mycelia) or other nascentspore structures and such mycelium should be considered a “preconidialmycelium” as discussed elsewhere.

It was found that the “fragrance signature” of the mycopesticidalmycelium is a strong attractant to insects prior to conidia formation.The genesis for these findings was the initial observation that the odorof the cultured mycelium was similarly pleasing to humans whenpreconidial and repellant after conidia formation; smell and thefragrance signatures of mycelium are utilized by the present inventor asindicators of the health of the mycelium in large scale production ofgourmet and medicinal mushrooms, whereas “petri dish mycologists” andentomologists studying pathogenic fungi are typically trained not tosniff or inhale from the cultures. It was noted such fragrancesignatures are lost when mycelium is grown via liquid fermentation—thismay be due to such fragrance signatures being “washed away” or due tothe greatly reduced nutritional base available to the mycelium in liquidfermentation as compared to solid substrates such as grain or wood, as“outgassing” of the mycelium of CO₂ and attractant molecules is believedby the present inventor to be responsible for at least some portion ofthe attractant value. It was also noted that liquid fermentationutilizing a typical fermentor with bubbled air mixing will promoteconidia formation, with such conidia production being even furtherpromoted by the common commercial practice of utilizing bubbled orchemically generated oxygen.

In addition to the attractant properties and phagostimulatory (feedingstimulating) properties of preconidial mycopesticides, it was furtherfound that pathogenic fungal control agents are much more effective whenpreconidial (pre-sporulation) mycopesticidal mycelium is ingested and/orcontacted by the targeted insect as compared to conidia orpost-sporulation mycelium/conidia offered to targeted insects for thepurpose of infection by contact. The preconidial mycopesticidal myceliumis thought to be an effective attractant and/or pathogen, at least inpart, because it is a preferred food, particularly for social insectsand other fungi-feeding insects

The preconidial mycelium has been observed to be a preferred food sourcewhich stimulates “grazing” of the fungi on wood and/or grain, scatteringof the fungus and caching of the fungus by social insects includingtermites, carpenter ants and fire ants. Novel behaviors observed in thesocial insects include that of Formosan termites (Coptotermesformosanus) ignoring available wood to set up “housekeeping” in themycelium and fire ants and carpenter ants moving the preconidial fungiaround the feeding arena and/or into nest chambers. Social insectcolonies have been described as “factory fortresses.” Wilson, E. O.,supra, and Oster, G. F. and E. O. Wilson, Caste and Ecology in theSocial Insects, Princeton University Press (1978). While it may bedifficult for a parasite to “break into the fortress” and gain access toa colony, once inside, the opportunities abound. Schmid-Hempel, supra,p. 77. Similarly, once the social insect defenses have been penetratedvia the attractiveness of preconidial mycopesticidal mycelium, theopportunities abound for further inoculation and spread of thepreconidial mycelium both orally and dermally, as well as optionalintroduction of other biocontrol agents or chemical toxicants. Novel andunique features of the invention include the use of a mycopesticidalmycelium or extract as an attractant, the use of a mycopesticidal vectorof parasitization that relies directly on hyphal fragments to infectboth insects and/or social insect housing structures, the use of highlevels of carbon dioxide to grow and maintain preconidial mycelium, theuse of late sporulating strains so as to prolong the attractivepreconidial state, the use of various methods to arrest development atthe preconidial stage and/or to facilitate growth, packaging, shippingand convenient application by an end user and various improvements inmethods of attracting, controlling, preventing, eradicating and limitingthe spread of insects.

Preconidial mycelium has proven to be highly effective by ingestion orcontact, the mycelial hyphae already being in a state of active growthwhen ingested or contacted. The preconidial mycelium is thought by thepresent inventor to function both as a “fungal food of infection” and asa contact insecticide. Efficacy as a contact insecticide is believed tobe aided by the somewhat “sticky” nature of mycelium. While not wishingto be bound by any theories or hypotheses, the present inventor believesvarious possible vectors for further spread and growth of thepreconidial mycelium include incidental contact and adhesion, feedingand “sloppy eating” which may spread hyphae to insect cuticles, foodcaching, individual and social grooming, aerial transmission of hyphalfragments (as dry hyphal fragments are much less dense than spores, theyeasily become airborne and spread), inhalation, incidental contact,trophallaxis (exchange of liquid food), proctodeal trophallaxis(exchange of anal excrement by termites and others), cannibalism,mating, contact with cadavers, inoculation of housing structures, etc.Mycopesticidal species are thought by the present inventor to employvarious pathogenic modes when transmitted via ingestion or contact withmycelial hyphae—for example, infection via the cuticle, the trachealopenings, the alimentary canal or wounds with resultant growth upon theinsect and resultant depletion of host resources and/or damage ordestruction of host tissue, production of antibiotics, antibacterialsand antiprotozoans with the resultant death of microflora within thegut, production of anti-fungal compounds affecting symbiotic andassociated fungi, production of toxic substances by the entomopathogen,suppression or disruption of the immune system response, etc. Themycelial hyphae may also inoculate the excavations and/or organic debrishousing structures (such as the termite mounds of wood or varying plantmaterials and debris cemented with salivary or fecal secretions) ofsocial insects. As the social insects disrupt and distribute theindividual particles of mycelium throughout the colony, the mycelium isselectively encouraged to continue to grow in a preconidial state,delaying the time to sporulation, as the fragments of hyphae re-growupon encountering new food sources. Hence another advantage to thisinvention is the further delaying of sporulation by the targeted insectcolony, thus insuring full inoculation of the nest.

Furthermore, whereas conidial preparations are more dependent uponhumidity in the insect environments, attractant preconidialmycopesticidal mycelium which is eaten or contacted by an insect isdependent in some part only upon the humidity in the immediate vicinityof the mycelium, the humidity of the mycelium of course being much moreeasily controlled than that in the wider general insect environment. Themicroenvironment “on” an insect is likely more conducive to hyphalgrowth than to spore germination, with hyphal growth typically beingmore immediate among fungi than spore germination. Optimal humidityconditions (often very high) and temperatures (depending on the type offungus) are necessary for germination, and the process is also sensitiveto the presence of secreted substances on the host's body surface, e.g.,fatty acids and other fungicidal substances. Schmid-Hempel, supra, p. 36and the references cited therein. Fungi may also enter through thealimentary tract as known from Beauveria bassiana in the fire antSolenopsis and in termites. However, the hyphae enter the body cavitysome 72 hours after ingestion of spores, and the chemical environment ofthe alimentary tract may be very aggressive and destroy the growinghyphae, although tracheal openings are an obvious target. Schmid-Hempel,supra, pp. 36, 43-44. Fungal spores are not always infective if ingestedby ants because of a filter apparatus established by the infrabuccalcavity and/or the proventriculus just in front of the stomach.Schmid-Hempel, p. 106. On the other hand, it has been recognized thatwith the exception of a few Entomophthorales, all entomopathogenicspecies can be cultured on artificial media, and are thereforefacultative rather than obligate parasites even though they may beobligate symbionts or pathogens in the ecological sense if they have nocapacity for free-living existence, other than as propagules, in theabsence of a suitable host. Keller, S. and K. Zimmermann, Mycopathogensof Soil Insects, in Wilding et al. (eds.), Insect-Fungus Interactions:14th Symposium of the Royal Entomological Society of London incollaboration with the British Mycological Society, Academic Press, pp.250-251 (1989). Although preliminary experiments reveal that M.anisopliae is a poor competitive saprophyte, (Zimmermann, supra,unpublished, evidently with spores), the present inventor has found bothMetarhizium and Beauveria to be facultative saprophytes that do quitewell on grain and wood in the preconidial stage when cultivated understandard sterile mushroom growing conditions. This saprophytic ability,coupled with the “mycelial momentum” the present inventor has observedin fungi in general, indicates that preconidial mycelium may thuspresent numerous advantages that have not been heretofore observed, suchas higher transmission rates and effectiveness toward targeted pestinsects where mortality has not been previously observed due to failureof spores to adhere and germinate or other virulence factors. Themycopesticidal life cycle stage of spore germination is skipped entirelyvia utilization of preconidial mycelium, with hyphal growth in or on aninsect commencing directly. Furthermore, as social insects spreadmycelium throughout the colony, nestmates and the feeding and nestingchambers may become inoculated. As the mycelium permeates thesurrounding habitat, resident moisture increases as a direct consequenceof the sponging and transporting properties of the mycelium. In doingso, more mycelium grows, increasing the effectiveness of this method. Asan additional benefit, the mycelium may dry out and become “reactivated”by humid conditions, thus functioning as a prophylactic and preventativetreatment and long-term protectant.

Because mycopesticidal mycelium as a food or ingested mycelium seems totake longer for parasitization by the fungi to become obvious to asocial insect colony as compared to spore germination on an externalsite, the time for the colony to recognize and isolate infectedindividuals is similarly prolonged, with the result of infectious spreadthroughout the colony before the defensive mechanisms of social insectscan react to infected individuals and maturing hyphae. As the delay isincreased, there is correspondingly less likelihood of learned responsesand quarantine of infested individuals or colony chambers.

It has been found by the present inventor that the preconidial stage canbe maintained in many mycopesticidal strains provided carbon dioxide(CO₂) levels are maintained above the approximately 365 ppm of carbondioxide currently found in the atmosphere, to with: at an elevatedlevel, e.g., 500 ppm. The CO₂ levels preferably range from 2,000 ppm,preferably 6,000 ppm, more preferably 10,000 ppm. The upperconcentration of CO₂ is preferably not more than 200,000 ppm, morepreferably not more than 100,000 ppm, and most preferably not more than50,000 ppm in air. Once exposed to fresh air, the mycelium of manystrains can produce conidia in just a few days. By preventing conidialformation, the mycelium continues to accumulate mycelial biomass (sansconidia). This prevention of conidia formation is a central component inthis technology, as conidia formation is thus actively avoided. Strainsthat do not sporulate under elevated carbon dioxide conditions afterovergrowth of the substrate for at least 1, and preferably 5 additionaldays after overgrowth are preferred, more preferably those that do notsporulate for 10 additional days, and most preferably those that do notsporulate for 60 days, or more. Thus, for example, Metarhiziumanisopliae #62176 has been observed to maintain a preconidial state inspawn bags with filter patches for 35 days or more after overgrowth, andBeauveria bassiana #20872 and 70438 have been observed to maintain apreconidial state for 60 days. Strains that do not sporulate for atleast three days after exposure of the preconidial mycelium to airand/or dirt are preferred, those strains that do not sporulate for atleast seven days are more preferred, while still more preferred arestrains that do not sporulate for at least 10 days, or most preferredare strains that do not sporulate for 21, or more, days. Thus, evenafter overgrowth of the substrate in the cultivation container, themycopesticidal mycelium may be maintained in a preconidial state viaelevated carbon dioxide levels and/or refrigeration at 1-10 degrees C.and maintained in a preconidial state after exposure to the elements.

Mycopesticidal mycelium is grown in pure culture using standardfermentation techniques well established for in vitro propagation ofmycelium from mycelium or spores. For example, the fermented mycelia isdiluted and transferred into a sterilized grain or a mixture ofsterilized grains (rice, wheat, rye, oat, millet, sorghum, corn, barley,etc.); alternatively, mycelia may be cultured on a petri dish (frommycelium or spores) and transferred to grain or other standardtechniques may be utilized. The grain is pressure steam-sterilized atone (1) kg/cm² (15 psi) for 30 minutes to several hours, depending uponprocessing parameters such as mass to be sterilized, type of autoclave,compartmentalization and other factors. The master broth is transferredaseptically manually or by using peristaltic pumps into the sterilizedgrain. Alternatively, growth mediums of or containing wood (includingbait chips and bait traps), sawdust and/or wood chips, agriculturalwastes, cardboard, paper, fiber blankets or other cellulose-containingsubstances may be utilized for cellulose loving insects (includingtermites and carpenter ants). A variety of containers are used forincubation, including high-density polyethylene and polypropylene bags,glass and polypropylene jars, metal containers, etc. Use of suchcontainers provides a convenient method of maintaining high carbondioxide levels, as the growing mycelium gives off CO₂. Carbon dioxidelevels will rise to acceptable levels for use in the present inventioneven if filter patches, disks or materials are utilized to allow somegas exchange. Alternatively, grow rooms may be maintained at high CO₂levels. Further information on such culture techniques, includinginformation of the selection and isolation of species, strains,varieties and sectors of cultures may be found in the applicant's books,Growing Gourmet and Medicinal Mushrooms, Ten Speed Press (1993, 2000)(Library of Congress Card Catalog Number SB353. S73 2000) and TheMushroom Cultivator, Agarikon Press, (1983) (with J. Chilton) (Libraryof Congress Card Catalog Number SB353. S74 1983), hereby incorporated byreference.

Once inoculated, the mycelia on grain (or on wood or other cellulosic,ligninic, celluloligninic or carbohydrate containing substrate or othernatural or artificial substrate) matures to a state prior to conidiaformation and may be utilized fresh or metabolically arrested ordevelopmentally arrested through flash chilling (freeze-drying), drying,refrigeration, cooling via nitrogen, carbon dioxide, or other gasses,absence of light, or control by other means. It will be understood thatsuch metabolic arresting of development may encompass either a slowingof metabolism and development (such as refrigeration) or a totalsuspension or shutdown of metabolism (freeze-drying, air-drying andcryogenic suspension). When freeze-drying, drying or other known methodsof arresting development are utilized, it is essential thatfreeze-drying or other methods occur at the stage in the life cycle ofthese fungi before the spores are produced. The mycelium-impregnatedgrain media may then be fragmented and packed in appropriate containersfor commerce. Fresh, dried and freeze-dried materials may alsooptionally be enhanced by use of protectants and nutrients (sugars areone preferred material that have both protectant and nutrientqualities), and materials such as wetting agents, surfactants andsurface active agents, dispersants, emulsifiers, tackifiers oradhesives, penetrants, fillers, carriers, antibiotics, germinationenhancers, growth enhancers, carbohydrates, nutritional supplements,spore and hyphae encapsulating materials, yeasts, bacteria, fungiperfecti and imperfecti, etc. Alternatively, fresh, dried orfreeze-dried preconidial material may be utilized within acellulose-containing or starch sheath or coating for enhancing theeffectiveness as a delivery system, and for attractingcellulose-consuming insects. Fresh mycelium may be shipped in growingcontainers such as jars or spawn bags, which allows easy maintenance ofa high carbon dioxide atmosphere and maintenance of sterile conditionsduring shipping.

When the freeze-dried or dried mycelium is reactivated via rehydration,the mycelium is typically preferably allowed to slowly rehydrate throughcontrolled absorption of atmospheric humidity, with the result that themycelium “wakes up” and wicks into the air. This is a very differentresponse from immersion, which often results in bacterial contaminationand souring, as the freeze-dried mycelium of some, but not all,mycopesticidal species suffers when immersed in water. Such rehydrationand reactivation may be carried out on a large scale through highhumidity atmosphere, or may be accomplished by an end user through useof wet materials such as sponges, wicking materials and/or otherevaporative materials or by atmospheric absorption of humidity from aremote water reservoir. Such end user rehydration may be carried out inany suitable container or a bait box if desired. Where species orstrains do not respond adversely to water saturation, immersion or othersaturation or partial saturation techniques may be utilized. Warming issuitable for reactivation of refrigerated materials; it is preferredthat the mycelium not be refrigerated for extended lengths of time.

Particularly preferred are those strains that are slow to sporulate(i.e., late conidia formers, thus prolonging the preconidial stage) bothbefore and after exposure to air and/or dirt that additionally possessthe desired characteristics of attractiveness to the targeted insect,virulence and pathogenicity (or lack thereof), low mortality rate ofnon-targeted insects, time to insect death, mortality rate for virulentstrains, the proportion of kill of each life stage of target insectssuch as larvae, pupae, workers, soldiers and royalty, high transmissionrates, host specificity for targeted insects, growth rate and speed ofcolonization of substrates, sensitivity and response to high and lowcarbon dioxide levels, recovery from drying, freeze-drying,refrigeration or other forms of metabolic arrest, recovery fromtransportation, stress tolerance, preferred temperature and humidityconditions matched to insect ecology and abiotic conditions, microflorasensitivity, ability to surpass competitors, adaptability to singlecomponent, formulated and complex substrates, high production ofattractant essences and extracts, genetic stability/instability,post-sporulation pathogenicity and virulence, etc. The interactionbetween mycopesticidal fungi and their insect hosts is complex anddynamic and no single trait or component of the invasion process islikely to determine virulence (or pathogenicity). Virulence also may bedetermined by environmental factors—as some mycopesticidal species areknown to become non-virulent when cultured on non-insect media, suchcultured species are likely to become more virulent as the preconidialmycelium spreads via social grooming, incidental contact, contact withcadavers, etc. Those skilled in the art will recognize that suchvirulence characteristics can be selected for utilizing known techniquesand bioassays.

As the preconidial or pre-sporulation mycelium is an attractant whereasthe conidia spores are more or less repellant, most preferred would be“sporeless” or “slow-to-sporulate” strains of mycopesticidal fungi.

To select an entomopathogenic species, strain or variety or sub-strain,or to select for a characteristic within those species, strains,varieties or sub-strains, or to isolate and propagate cultures involveschoices that affect the downstream methods, products and activities.Such selection might involve location and isolation of a species orstrain from the wild or the acquisition from a culture bank or othersource, the selection of a petri dish culture which manifests thedesired characteristics, the selection of a strain which gives favorableresults in a bioassay or the selection of a sector within a petri dishculture for healthy growth or other characteristics. If selecting forslow to sporulate strains or sub-strains, one would choose to cultureout those sectors displaying the preconidial mycelium rather than thosesectors displaying post-conidial mycelium.

More than one fungus and/or substrate can be used to create a matrix ofcharacteristics to increase usefulness as a natural pesticide andmaximize effectiveness against an individual or a diverse population ofeconomically or ecologically damaging insect species. For example, aplurality of Beauveria, Metarhizium, Paecilomyces, Verticillium, and/orCordyceps species and strains might be utilized, a mixture of grains ofvarious sizes might be utilized and/or a mixture of various grain andcellulosic substrates might be utilized. Grains may be selected based onsuch factors as an insect's mandible size, preferred food particle size,insect size and size of grains that the insect may grasp and/or carry,insect preference, similarity to pupae, etc. Woods and the wide varietyof cellulosic materials may similarly be selected based on insectpreferences; for example, birch and pine are preferred woods of manyinsects. Attractant properties may vary between species and with eachstrain, also affected by type of grain or carrier material (wood blocks)and various other factors, and mycopesticidal strains may be selectedfor peak attractant properties on particular cellulosic substrates aswell as attractant properties in general.

In utilizing wood and other cellulose containing materials, onepreferred method is to grow the pre-sporulation mycopesticidal myceliumon wooden or other cellulosic materials “bait blocks” or “bait traps.”Bait chips, blocks or traps (or optionally other forms such as pellets,extruded pellets, mats, fabrics, ropes, etc.), optionally soaked with amalt solution or other sugar and/or nutrient solution, are infusedand/or inoculated with preconidial mycopesticidal mycelia which thenspread the infection to the targeted insect pests via any of themycelium vectors described herein. Biodegradable bait traps may be madeof, or have components made of, various cellulosic, ligninic,celluloligninic, carbohydrate and fiber materials including but notlimited to paper products and cardboard, wood and sawdust, corn cobs andcornstalks, chip board, jute, flax, sisal, reeds, grasses, bamboo,papyrus, coconut fibers, nut casings such as peanuts, almonds, walnuts,sunflower, pecans, etc., seed hulls such as cottonseed hulls, hemp,cereal straws, sugar cane bagasse, soybean roughage, coffee wastes, teawastes, cactus wastes, banana fronds, palm leaves, fiberized rag stock,combinations thereof, and numerous other forest and agriculturalproducts and byproducts which will host mycelium and are degradable bymycopesticidal fungi. Where rapid biodegradability of the traps isdesired, materials such as cardboard or paper may be utilized. Forinsects including carpenter ants or termites, cockroaches, etc., thebait blocks preferably contain channels, tunnels, grooves, ridges,holes, or perforations specifically sized to allow entry by the targetedspecies and or its brood, pupae and/or larvae. Inoculation may, forexample, be accomplished via grain in the channels and the blocks mayoptionally be layered or “wafered” together. A composite, layered orintertwined matrix of materials may be utilized, with one set ofmaterials infused with the attractant extract of an entomopathogenicspecies and the other containing active or metabolically arrestedpreconidial mycelium. A multiplicity of such bait blocks or traps orbarriers may be utilized to protect structures, agricultural locations,etc. A fungal matrix with a plurality of pre-sporulating mycopesticidalfungal species and/or extracts that are highly attractant to thetargeted pest insect may be created so that the targeted pest is drawnclose to a locus where the insect pest becomes infected and is harmed orkilled by the selected fungi or via other means.

The wooden or cellulose baits and bait traps may optionally be dried orfreeze-dried. Either the myceliated bait may be presented to the insect,with rehydration and recovery taking place, for example, within thecentral nests of social insects, or the wooden bait block may berehydrated prior to or during use.

The highly attractive nature of preconidial mycopesticidal myceliumindicates that essences extracted from preconidial mycelium ofmycopesticidal fungi can be expected to be highly attractive in and ofthemselves, and thereby similarly useful alone or in conjunction withbiological, chemical, mechanical and/or electronic insect controlagents, useful as masking agents for otherwise repellant toxicants forinsect pests, and useful as “distractants” in diverting insects awayfrom sites that need protection. Such essences include extracts,concentrates, fragrances, derivatives, active constituents, etc. and maybe prepared by methods known to the art including extraction with water,alcohols, organic solvents and supercritical fluids such as CO₂, etc.Extracts may also be prepared via steam distillation of volatilecomponents, similar to the preparation of “essential oils” from flowersand herbs. Suitable alcohols include those containing from 1 to 10carbon atoms, such as, for example, methanol, ethanol, isopropanol,n-propanol, n-butanol, 2-butanol, 2-methyl-1-propanol (t-butanol),ethylene glycol, glycerol, etc. Suitable organic solvents includeunsubstituted organic solvents containing from 1 to 16 carbon atoms suchas alkanes containing from 1 to 16 carbon atoms, alkenes containing from2 to 16 carbon atoms, alkynes containing from 2 to 16 carbon atoms andaromatic compounds containing from 5 to 14 carbon atoms, for example,benzene, cyclohexane, cyclopentane, methylcyclohexane, pentanes,hexanes, heptanes, 2,2,4-trimethylpentane, toluene, xylenes, etc.,ketones containing from 3 to 13 carbon atoms such as, for example,acetone, 2-butanone, 3-pentanone, 4-methyl-2-pentanone, etc., etherscontaining from 2 to 15 carbon atoms such as such as t-butyl methylether, 1,4-dioxane, diethyl ether, tetrahydrofuran, etc., esterscontaining from 2 to 18 carbon atoms such as, for example, methylformate, ethyl acetate and butyl acetate, nitriles containing from 2 to12 carbon atoms such as, for example acetonitrile, proprionitrile,benzonitrile, etc., amides containing from 1 to 15 carbon atoms such as,for example, formamide, N,N-dimethylformamide, N,N-dimethylacetamide,amines and nitrogen-containing heterocycles containing from 1 to 10carbon atoms such as pyrrolidine, 1-methyl-2-pyrrolidinone, pyridine,etc., halogen substituted organic solvents containing from 1 to 14carbon atoms such as, for example, bromotrichloromethane, carbontetrachloride, chlorobenzene, chloroform, 1,2-dichloroethane,dichloromethane, 1-chlorobutane, trichloroethylene, tetrachloroethylene,1,2-dichlorobenzene, 1,2,4-trichlorobenzene,1,1,2-trichlorotrifluoroethane, etc., alkoxy, aryloxy, cycloalkyl, aryl,alkaryl and aralkyl substituted organic solvents containing from 3 to 13carbon atoms such as, for example, 2-butoxyethanol, 2-ethoxyethanol,ethylene glycol dimethyl ether, 2-methoxyethanol, 2-methoxyethyl ether,2-ethoxyethyl ether, etc., acids containing from 1 to 10 carbon atomssuch as acetic acid, trifluoroacetic acid, etc., carbon disulfide,methyl sulfoxide, nitromethane and combinations thereof. Extracts mayalso be prepared via sequential extraction with any combination of theabove solvents. The extracts may optionally be combined with fixatives,enhancing agents, oils, alcohols, solvents, glycerin, water and othersubstances that aid in distributing the attractant and/or enhancing itsfragrance value. Essences extracted from preconidial mycelium ofmycopesticidal fungi can be used as a protectant or distractant, luringinsects away from a locus and preventing insect damage to a locus,habitat, structure, crop, animal, human, etc. Such attractant essencesand extracts may be utilized with wicking agents, sprayers, etc. toenhance their effectiveness. Preliminary indications are that suchattractant molecules are polar and thus best extracted with polar and/orhydrophilic solvents. The present invention in conjunction with theprinciples of chemical ecology and evolutionary biology raise thepossibility that the entomopathogenic fungal species produce attractantmolecules (or more likely, groups of attractant molecules) that havecoevolved over evolutionary time with species of insects or groups ofinsects. Such attractant molecules, optimized for one species of insect,may well show attractant properties to larger groups of insects. It willbe apparent to those skilled in the art that numerous such molecules orgroups of attractant molecules may be isolated and/or characterized fromthe preconidial fungi of the present invention and as such should beconsidered part of the present invention.

The preconidial mycelium or extracts thereof may be utilized solely asan attractant for various purposes. For example, preconidial myceliummay be utilized to affect insect choice of geographical location,destructive pests being attracted and distracted away from structures,agricultural plots, etc. Fungal species and strains particularlyattractive to beneficial insects may be utilized to attract desiredinsect species, the fungi acting as a biological catalyst to steer thecourse of the insect community evolution. Alternatively, varying insectsmay simply be attracted to occupy the environment and thus forestallpest invasions. It is known that virulence of entomopathogenic strainsvaries widely in the laboratory when tested via typical conidia basedassays, with mortalities from 0% to 100% being recorded dependent uponsuch factors as number of conidia applied per insect and the insectspecies and the entomopathogenic species and strain being tested (see,for example, the U.S. patents above). Similar results may be expectedfor preconidial formulations, although a greater effectiveness ingeneral may be expected since lack of virulence in the typical bioassayis often related to a failure of conidia to adhere to the insect and/orfailure of the conidia to germinate as discussed above. Thus strains of“pathogenic” or “entomopathogenic” fungal species may be selected whichactually vary in virulence from non-pathogenic to relatively weaklyvirulent to strongly virulent. Non-virulent preconidial mycelium may beused to attract beneficial predator and parasitic insects.Alternatively, non-virulent strains may be utilized as a distractant,for example attracting Coccinellidae, the lady beetles, away from areaswhere they may be a pest (such as office buildings) and into “ladybugmotels.” Alternatively, virulent strains may be utilized as an olfactoryattractant but made inaccessible with devices such as screens or slots.

The mycoattractants and/or mycopesticides disclosed herein may also beoptionally enhanced by the use of other baits, foods, attractants,arrestants, feeding stimulants, sex pheromones, aggregating pheromones,trail pheromones, etc. For example, a bait box overgrown withpreconidial mycopesticidal mycelium might contain other attractants andcontact pesticides.

Attractant preconidial or pre-sporulation mycelium (virulent, weaklyvirulent and/or non-virulent) or extracts may also be utilized inconjunction with other biological organisms, chemical pesticides andphysical control agents as part of integrated pest management (IPM)systems that incorporate multiple pest control tools and seek tominimize pesticide inputs. The use of attractant fungi in combinationwith other insect control agents affords the advantage of attracting thetargeted pest to a locus which, by other treatments, results insterility and/or death of the targeted insect.

The weakened immune systems of pest insects exposed to pathogenic orvirulent mycopesticidal organisms allows other beneficial parasitic andpredator species to flourish. Such beneficial biological control agentsinclude microbial pathogens, predator insects (entomophagous insectswhich eat other insects) and parasitic insects (those which reproduce bylaying eggs in or on any stage the host insect, from egg to adult), aswell as non-insect predators such as birds and beneficial nematodes,spiders and mites. Examples of biological control agents includeentomopathogenic fungal species and their spores, Bacillusthuringiensis, B. popilliae and B. subtillis and Pseudomonas, fire antparasites (such as Phorid flies), fly parasites (wasps such asMuscidifurax raptorellus and Spalangia camerons), hister beetles such asCarcinops pumilio, dung beetles including Onthophagus spp., parasiticnematodes such as Steinemema feltiae, cockroach parasites (Anastatustenuipes, Aprostocetus hagenowii, Comperia merceti and nematodes),lacewings, ladybugs, bigeyed bugs, damsel bugs, praying mantises,Trichogramma wasps, beneficial mites, ant parasites, flea parasites,lygus bug parasites, mealybug, aphid and whitefly parasites andpredators, caterpillar parasites, spider mite predators, looperparasites, diamondback and moth parasites, scale parasites andpredators, mite parasites and predators, etc. Strains may be selected,utilizing those methods known to the art, for virulence against thetargeted pest insects and/or non-virulence or weak virulence againstpredator insect species as well as such qualities as resistance topesticides, etc. If desired, resistant predator or parasitic species maybe selected for, bred and released to further control the targeted pestspecies. Blends of beneficial insect attractant plants and habitatplants may also be utilized. This dualistic approach is not limited tojust one pairing of fungus and beneficial organism as many pairingscould be implemented for the purpose of creating an environmentalequilibrium affording long-term protection. Other fungal attractants mayalso be optionally utilized. Thus, a combination of the preconidialmycelium of mycopesticidal species and Oyster mushrooms (Pleurotus andHypsizygus species, the mycelium and mushrooms of which are veryattractive to Phorid flies) might be utilized to attract phorid flies inthe genus Pseudacteon that parasitize fire ants and leaf-cutter ants.

The preconidial mycopesticides (both virulent and non-virulent strains)and extracts may also be utilized as “masking agents” as well asattractants in conjunction with insect chemical control agents,toxicants and/or pesticides, thereby preventing aversion to othereffective compounds that may otherwise repel the insect. Chemicalcontrol agents include insect toxicants, poisons, regulators andpesticides as well as the chemicals (semiochemicals) which mediateinteractions between individuals of a insect species (pheromones) orbetween co-evolved species (allelochemicals, such as kairomones andallomones). Residual (persistent), non-residual (nonpersistent) andsolid, liquid, aerosol or fog contact chemical control agents include,by way of example but not of limitation, stomach poisons such assulfluramid, pyrethrum extracts, natural and synthetic pyrethroids,parapyrethroids (non-ester pyrethroids) such as silafluofen, etofenproxand cyfluthrin, pyrethroid analogs such as fenvalerate, permethrin,phenproparthrin, fluvalinate, flucythrinate, fenproparthrin,cypermethrin, deltamethrin, tralomethrin, cycloprothrin, esfenvalerateand zeta-cypermethrin, allethrins, lethanes, nicotinyl compounds such asimidacloprid, phenylpyrazoles such as fipronil, amidinohydrazones suchas hydramethylnon (a respiratory poison), abamectin (a mixture ofavermectins, insecticidal or anthelmintic compounds derived from thesoil bacterium Streptomyces avermitilis), Spinosad (spinosyn metabolitesproduced by S. spinosa), nitromethylenes, carbamates such as propoxurand fenoxycarb, organophosphates such as acephate and chlorpyrifos,pyriproxyfen, insect growth regulators, synthesis inhibitors, chitinsynthesis inhibitors such as hexaflumuron and diflubenzuron, mineralacids such as boric acid, alcohols and organic solvents, elements suchas sulfur and combinations thereof. Such chemical control agents mayoptionally be combined with synergists compounds that increase thetoxicity and/or enhance the biological activity of another, for exampleby inhibiting the enzymatic detoxification of insecticides by microsomaloxidases or hydrolytic enzymes such as esterases. Examples of synergistsinclude methylenedioxyphenyl (MDP) compounds such as piperonyl butoxide,piperonal bis-(2,2-(butoxyethoxy)-ethyl)acetal,1,2-methylenedioxynaphthalene, tropital (polyalkoxy acetal ofpiperonaldehyde) and sesamex, trisubstituted aliphatic and aromaticphosphates such as TOCP (tri-o-cresyl phosphate), a number ofnon-insecticidal carbamates, EPN(O-ethyl-O-p-nitrophenylphenylphosphonothionate), sulfoxide, propynyl ethers, p-nitrobenzylthiocyanate, 2-((4,6-dichloro-2-biphenylyl)-oxy) triethylamine,2-(diethylamino)ethyl 2,2-diphenyl pentanoate, 2-propynyl4-chloro-2-nitrophenyl ether, N-octyl bicycloheptane dicarboximide andn-propyl isome. Use of attractant or attractant/pesticidal preconidialmycelium or extracts enables the use of extremely small amounts oftoxicant or pesticide to effectively control insect populations.Alternatively, sublethal doses of pesticides or toxicants may beincluded to enhance the activity and virulence of the mycopesticidalspecies; or pathogenic and virulent preconidial mycelium may be utilizedas a preconditioning treatment, increasing the susceptibility to and/orpotentiating the virulence of other agents (such as pesticidalchemicals, other mycopesticides, or bacteriological, plasmodial andviral compounds). Lethal or sublethal doses of insect toxic materialsmay optionally be encapsulated within an attractant extract- ormycelia-impregnated (virulent or non-virulent) sheath, coating,covering, encapsulative material, protective and/or time degradingenvelope, or the toxin may surround, cover or encapsulate a mycelialsubstance or extract of strong attractive and/or mycopesticidalproperties, or such may be simply mixed.

The mycoattractants and mycopesticides of the present invention may alsobe combined with physical control agents. Physical control agents aredevices that destroy insects directly or act indirectly as barriers,excluders, or collectors. Physical controls include the use ofmechanical and electrical devices, heat, light, electricity, X-rays, andso on, to kill insects directly, reduce their reproductive capacity, orto attract them to something that will kill them. Various physical meansmay be employed to act as barriers to insect movement. Sticky materialsin which insects become hopelessly entangled may be used in the form offlypaper or coated objects and materials. Traps may be used for control,survey, and surveillance purposes. Control traps may be used inconjunction with mycoattractants and with some means of killing theinsects that enter (e.g., a pesticide or an electrically charged grid).

The preconidial mycelium on manufactured, compressed pellets orgranules, with or without additional liquid(s), can be used forapplications in agricultural, forest, industrial and/or domesticsettings, wherein the myceliated pellets become a loci for attractingthe target pests, and thus through contact become infected. Trends inmushroom spawn for gourmet and bioremediation purposes have long beenevolving towards pelletized or granular spawn while mycopesticidal sporetechnology similarly has evolved toward granulated or sprayformulations. Various forms of pelletized spawn, coated compositions,granules and dusts are known, including those formed from nutrients,with or without carriers and binders, such as peat moss, vermiculite,alginate gel, wheat bran, calcium salts, hydrophilic materials such ashydrogel, perlite, diatomaceous earth, mineral wool, clay, polymers,biopolymers and starch, including wettable powders, emulsifiableconcentrates, starch and/or biopolymer coatings, etc. Pelletized spawnis specifically designed to accelerate the colonization processsubsequent to inoculation. Idealized pelletized spawn seeks a balancebetween surface area, nutritional content, and gas exchange and enableseasy dispersal of mycelium throughout the substrate, quick recovery fromthe concussion of inoculation, and sustained growth of myceliumsufficient to fully colonize the substrate. See Stamets, supra, pp.141-142 and U.S. Pat. Nos. 4,551,165 (1985), 4,668,512 (1987), 4,724,147(1988), 4,818,530 (1989), 5,068,105 (1991), 5,786,188 (1998) and6,143,549 (2000). Liquid sprays include the above wettable powders andemulsifiable concentrates, water-dispersible granules, aqueoussolutions, emulsions such as oil-in-water and water-in-oil emulsions,dispersions, suspoemulsions, microemulsions, microcapsules, etc.Wettable powders are formulations which are typically uniformlydispersible in water and also contain surface active agents(surfactants) such as wetting agents, emulsifiers and dispersing agents.Emulsifiable concentrates are prepared with organic solvents and/or oneor more emulsifiers. Sticking agents such as oils, gelatin, gums,tackifiers and adhesives may be used to improve the adhesion of thespray. Humectants may also be used to decrease the rate of evaporation,including for example glycols having from 3 to 10 carbon atoms andglycerin and solutes such as salts or sugars in water.

The preconidial mycopesticidal mycelia of the current invention may alsobe applied as a protectant for equipment. For example, mycopesticidalmycelium may be grown on an organic or organic/synthetic covering suchas a sheath or membrane made with a matrix of organic materials such aspaper, cardboard, hemp, agricultural fibers, wood, etc., with or withoutnon-degradable materials, and utilized fresh or dried as appropriate.Such mycopesticidal sheaths may be utilized as a preventative barrier toprotect electrical cables and wires, computer cables, telephone wires,microwave equipment, optical networks, etc. from damage by fire ants,which can be attracted by electrical activity. Such mycopesticidalcoverings in conduits, ducts, corridors, etc. could be activated bydecreasing air flow and/or increasing humidity, depending onapplication, thus allowing dried mycelium to rehydrate and “reawaken” soas to deal with insect outbreaks. Such a solution might have helped savethe now-abandoned Superconducting SuperCollider project in Texas fromthe devastation caused by fire ants that damaged the electrical wiring.

For large scale application, fabric or fiber cloths, landscaping cloths,geofabrics, soil blankets and rugs, mats, mattings, bags, gabions, fiberlogs, fiber bricks, fiber ropes, nettings, felts, tatamis, bags,baskets, etc. made of biodegradable materials infused with preconidialmycelia of mycopesticidal species may be utilized as a mechanism forattracting, preventing, killing or limiting the spread of targetedinsects (or of attracting beneficial insects). Thus, for example,barriers or “aprons” of mycopesticidal mycelium grown on straw, coconutfiber, wood, paper, cardboard or the other forestry and agriculturalproducts, wastes and cellulose sources noted above might be placedaround Oak trees to protect from beetles and introduced wilts such asPhytophthora and Ceratocystis fagacearum or around pine trees or standsto protect from destructive fungi carried by bark beetles. Similarly,such mycopesticidal aprons might be utilized to protect other trees,shrubs, grasslands, rivers and streams, estuaries, riparian zones,agricultural fields, gardens and crops, structures, communities,habitats and sensitive ecosystems. Such preconidial mycopesticidalaprons might alternatively be used to attract pest insects to a sitewhereupon other biological, chemical, mechanical, electrical and/orother insect reducing treatments become more effective. Conversely,creation of buffers utilizing non-virulent strains selected forattractiveness to beneficial insects can be used to attract beneficialspecies which naturally parasitize problem insects.

Alternatively, woodchips, grains, hydromulch and other substratesinfused with preconidial mycelium may be utilized in spray hydroseedersor mobile hydroseeders. Agricultural equipment may be utilized toinoculate fields and agricultural wastes. The mycopesticidal fungi mayalso optionally be utilized in conjunction with saprophytic fungi andmycorrhizal fungi to enhance soils and agricultural yields (“companioncultivation” of beneficial fungi). Mycopesticidal species are alsouseful in the mycoremediation (fungal bioremediation) of various sites.As one example, reclaimed logging roads could become perimeter-barrierswhich could forestall and/or prevent beetle-plagues from devastatingforestlands by infusing mycomats or hydromulches with species-specificpathogenic fungi (and optionally saprophytic and mycorrhizal fungi),while simultaneously retaining other benefits of mycofiltration. Thus,mycopesticidal species such as Metarhizium, Beauveria and Cordyceps,mycorrhizal mycopesticidal fungi such as Laccaria, and myconematicidalsaprophytic fungi such as Pleurotus might be combined withectomycorrhizal and endomycorrhizal species and saprophytic fungi toprovide simultaneous insect control, road reclamation and protection ofstreams from silt runoff. As Hypholoma capnoides, a premier wood chipdecomposer, mycelium has been observed to be repellant to insects,stretches of insect repellant barriers may be combined with attractantmycopesticidal kill and/or control zones for insects such as wood-boringbeetles. Similarly, control of agricultural runoff utilizing saprophyticfungi on agricultural wastes might be combined with the presentmycoattractant and/or mycopesticidal applications.

Another approach may optionally be taken with the removal of diseasedtrees via utilizing chain saw oil as a carrier for mycopesticidalspecies. In this particular application, conidia or a mycelium/conidiamixture may be utilized as well as preconidial mycelium. Spores and/ormycelium of mycopesticidal fungi, for example Metarhizium, Beauveriaand/or Cordyceps, are infused into chain saw oils so that when theinfected trees are cut for removal, the remaining stumpage is inoculatedwith the mycopesticidal spores and/or hyphae, thus preventing orlessening insect invasions. As another example, a similar approach couldbe utilized to create “mycopesticidal barriers” in forested lands whereboring beetles are a major cause of disease. Such spored or hyphal oilsmay also be employed in applications such as ecological rehabilitationand mycoremediation. Chainsaw lubricants (“bar and chain” oil) andlubricating oils suitable for the practice of the present inventioninclude petroleum and mineral oil lubricants, including natural brightstock and neutral stock oils, synthetic or semi-synthetic oils,vegetable lubricants and modified vegetable lubricants, animallubricants and blends and combinations thereof, with or without additivepackages. Suitable commercially available biodegradable lubricantsinclude STIHL® BIOPLUS bar and chain oil (natural oils in combinationwith natural polymers, 99% vegetable-based canola oil), CASTROL® BIOLUBE100, and vegetable oil lubricants such as those disclosed in U.S. Pat.No. 5,888,947 to Lambert et al., herein incorporated by reference. Ingeneral, where oils are utilized, biodegradable oils are preferred asoffering a more readily available nutritional source to a wide varietyof fungi. The fungal hyphae or spores may optionally be supplementedwith further amendments including germination enhancers, growthenhancers, sugars, nutritional supplements, surface active and wettingagents, spore and hyphae encapsulating materials, yeasts, bacteria,fungi perfecti and imperfecti, etc. Fungal hyphal mass can optionally bedried or freeze-dried and packaged, with or without additional spores,in spoilage-proof containers for marketing to end users as an additive.Fresh mycelial hyphae or mycelial mass is best used immediately ratherthan stored for long periods.

By adding spores of mushroom fungi such as Hypholoma capnoides or otherinsect-repulsive species into chain saw oils, the resultant myceliumgrowing through the cuts in the dead wood repels pine beetles and otherboring insects, thus limiting further infection. By using attractantmycopesticidal species in conjunction with repellant fungal species inanother area, a selective influence can be exerted on which insectspecies can be brought to any locus or repelled from it.

In general, preferred mycopesticidal species as pathogens are somewhatslow-acting (that is, not immediately fatal) so as to avoid bait shynessand to avoid learning effects in social insects before individuals havedistributed mycelium to all other members of the colony. To effectcontrol of Coptotermes formosanus colonies, bait chemicals must killslowly enough to allow foraging termites to return to the colony andspread the toxin to other colony members. Wright et al., Growth responseof Metarhizium anisopliae to two Formosan subterranean termite nestvolatiles, naphthalene and fenchone, Mycologia, 92(1): pp. 42-45 (2000)and the references therein. Bait shyness and other colony defensemechanisms such as segregation or avoidance of infected nestmates ornecrophoretic behavior by the workers (i.e., removal of dead nestmates)serve as a means of defense against the spread of such pathogens whenthe targeted insect dies too quickly. For example, in general, queenfire ants will not feed on new foodstuffs until the food is firstsampled by foragers or workers or members of expendable classes anddeemed safe after a two or three day waiting period. Note, however, thisgeneral pattern may not always apply to the highly attractivemycoattractants and mycoattractants disclosed herein. Preconidialmycelium strains may be selected for virulence after an appropriate timeperiod. In many applications it may be preferable to utilize a mixtureor matrix of several species or strains of entomopathogenic fungus withdifferent characteristics, maturation and growth rates including strainswith delayed sporulation (and thereby prolonged attractant value) whilein other applications a single species may be preferred. Similarly, withreference to a single species, a mixture of strains or a single strainmay be utilized. A mixture of species and/or strains both allows thetargeted insects to choose the species to which they are most attractedand provides for the possibility of simultaneous infection and insectplagues from multiple virulent species and strains.

Those skilled in the art will recognize that numerous entomogenous andentomopathogenic fungal species are known to the art and the abovepreconidial mycoattractant and mycopesticidal methods and products maybe favorably applied to many or all such species, and it is the intentof the inventor that the invention be understood to cover such. Suitableentomopathogenic fungi include the Deuteromycetes Metarhizium,Beauveria, Paecilomyces, Hirsutella, Verticillium, Culicinomyces,Nomuraea, Aspergillus and other fungi imperfecti, sexually reproducingfungi such as the Ascomycetes Cordyceps, Ascosphaera, Torrubiella,Hypocrella and its Aschersonia anamorph, and the PyrenomyceteLaboulbenia hageni, the Basidiomycetes such as Laccaria, andcombinations thereof. The Entomophthoracae including Entomophaga,Massospora, Neozygites, Zoophthora, Pandora and other Phycomycetes arealso considered to be within the scope of the invention. Also includedare such entomopathogenic species that have been genetically modified tobe more virulent (including those modified via mutagenesis,hybridization and recombinant DNA techniques).

By way of example, but not of limitation, mycopesticidal species includeMetarhizium anisopliae (“green muscarine”), Metarhizium flaviride,Beauveria bassiana (“white muscarine”), Beauveria brongniartii,Paecilomyces farinosus, Paecilomyces fumosoroseus, Verticillium lecanii,Hirsutella citriformis, Hirsutella thompsoni, Aschersonia aleyrodis,Entomophaga grylli, Entomophaga maimaiga, Entomophaga muscae,Entomophaga praxibulli, Entomophthora plutellae, Zoophthora radicans,Neozygites floridana, Nomuraea rileyi, Pandora neoaphidis, Tolypocladiumcylindrosporum, Culicinomyces clavosporus and Lagenidium giganteum, thewide variety of Cordyceps and its imperfect forms including Cordycepsvariabilis, Cordyceps facis, Cordyceps subsessilis, Cordycepsmyrmecophila, Cordyceps sphecocephala, Cordyceps entomorrhiza, Cordycepsgracilis, Cordyceps militaris, Cordyceps washingtonensis, Cordycepsmelolanthae, Cordyceps ravenelii, Cordyceps unilateralis, Cordycepssinensis and Cordyceps clavulata, and mycorrhizal species such asLaccaria bicolor. Other mycopesticidal species will be apparent to thoseskilled in the art.

The concepts of “preconidial” and “spores” or “conidia” are complex,containing a number of different forms and specialized structures forreproduction of the fungi. Many fungi are pleomorphic, that is, onefungus may produce several sorts of spores which may or may not becoincident in time. With regard to the sexually reproducing Cordyceps,Laccaria and other “fungi perfecti,” preconidial or pre-sporulationrefers to the pre-fruiting state. The term “preconidial” or“pre-sporulation” has a somewhat different meaning with regard to thesexually reproducing fungi than with most other entomopathogenic fungi,as sexually reproducing fungi are “fungi perfecti” or mushroom fungi,whereas the non-mushroom fungi such as Beauveria and Metarhizium are themore primitive “fungi imperfecti.” The situation is complicated by thefact that entomophthoralean fungi have complex life cycles involvingnon-sexual conidia and sexual resting spores. The situation is furthercomplicated by the fact that some or all Cordyceps fungi are dimorphicand have a teleomorph (the sexual perfect form or morph, e.g. thatcharacterized by sexual spores including acospores and basidiospores)and one or more anamorphs (the asexual imperfect form or morph, e.g.characterized by the presence or absence of conidia) with conidialstages within the imperfect fungal genera including Beauveria,Metarhizium, Paecilomyces, Hirsutella, Verticillium, Aspergillus,Akanthomyces, Desmidiospora, Hymenostilbe, Mariannaea, Nomuraea,Paraisaria, Tolypocladium, Spicaria (=Isaria) and Botrytis. For example,C. subsessillis is the perfect form of Tolypocladium inflatum, ananamorph (imperfect) form which produces cyclosporin. Hodge et al.,Mycologia 88(5): 715-719 (1996). Cordyceps militaris (Fr.) Lk. is alsothought to be dimorphic, the conidial stage of which is believed to be aCephalosporium. Cordyceps unilateralis seems specific on theCamponotinii, while Hirsutella sporodochialis is probably an anamorph ofC. unilateralis specific on Polyrhachis. Schmid-Hempel, supra, p. 43.DNA studies are expected to better elucidate these relationships. Asused herein, unless otherwise specified, preconidial or pre-sporulationmycelium of sexually reproducing fungi refers to the pre-sporulationmycelial stage of the mushrooms, including any preconidial imperfectstages, but not any conidia bearing imperfect stages.

The “truly” social insects, or “eusocial” insects, include all of theants, all of the termites and the more highly organized bees and wasps.These insects can be distinguished as a group by their common possessionof three traits: (1) individuals of the same species cooperate in caringfor the young; (2) there is a reproductive division of labor, with moreor less sterile individuals working on behalf of fecund nestmates; and(3) there is an overlap of at least two generations in life stagescapable of contributing to colony labor, so that offspring assistparents during some period of their life. Social pest insects are aparticularly apt target for mycelial hyphae based control agents, as themycelium may contact numerous individuals of varying castes and mayinfest housing structures. For example, Metarhizium anisopliae seems toattack only queens of Solenopsis in South America (“queen's disease);however, this may only look like a queen pathogen because infectedworkers leave the nest and are never found. Schmid-Hempel, supra, p.110. Based upon observations to date and what is known to those skilledin the sciences concerned with entomology and entomopathogenic fungi, itis expected that preconidial mycoattractant and mycopesticidal productsand methods may be similarly developed and applied favorably to all pestinsects, including both social and non-social insects, and it is theintent of the inventor that the invention be understood to cover such.

Such mycoattractant and mycopesticidal preconidial fungi and extractsthereof are individually and/or collectively useful against such insects(Insecta) as termites (Isoptera) including Rhinotermitidae,Kalotermitidae, Termitidae, Mastotermitidae, Hodotermitidae andSerritermitidae such as subterranean termites, drywood termites,harvester termites, dampwood termites, desert termites and rottenwoodtermites, etc., including Coptotermes formosanus Shiraki (Formosantermite), Reticulitermes (e.g., R. flavipes, R. virginicus, R. speratus,R. hesperus, R. tibialis, R. lucifugus, R. santonensis), Cryptotermes(e.g. C. domesticus and C. cubioceps), Acanthotermes, Ahamitermes,Allodontermes, Amitermes, Amitermitinae, Anacanthotermes,Archotermopsis, Armitermes, Calcaritermes, Capritemmes, Cornitermes,Cubitermes, Drepanotermes, Globitermes, Glyptotermes, Heterotermes,Hodotermes, Hodotermopsis, Incisitermes (e.g. I. minor), Kalotermes(e.g., K. flavicollis), Labiotermes, Macrotermes, Macrotermitinae,Marginitermes, Mastotermes (M. darwiniensis), Microcerotermes,Microhodotermes, Nasutitermes, Nasutitermitinae, Neotermes,Odontotermes, Ophiotermes, Paraneotermes, Parastylotermes,Parrhinotermes, Pericapritermes, Porotermes, Prorhinotermes,Psammotermes, Rhinotermes, Rhynchotermes, Rugitermes, Schedorhinotermes,Serritermes, Stolotermes, Syntermes, Termes, Termitinae, Termitogeton,Termopsis and Zootermopsis, and ants, wasps and bees (Hymenoptera)including, for example, ants (Formicoidea: Formicidae) such as thecarpenter ants (Camponotini) Camponotus modoc, C. vicinus, C.ferrugineus, C. floridanus, C. pennsylvanicus, C. herculeanus, C.varigatus, C. abdominalis and C. noveboracensis, Calomyrmex, Opisthopsisand Polyrhachis, fire ants Solenopsis invicta and S. richteri, pharaohants (Monomorium pharonis), Argentine ants, pavement ants, odorous houseants and leaf cutter ants (Atta and Acromyrmex), wasps (Sphecoidea andVespoidea) and bees (Apoidea including the Apidae “killer bees” Apismellifera adansonii and Apis mellifera scutellata and hybrids thereof,particularly those hybrids with the European honey bee).

Just as the social insects have complex relationships with fungi in thewild, wood-boring beetles have intimate relationships with “ambrosiafungi” and other fungi as preferred food sources. It is expected thatthe preconidial mycopesticidal products and methods disclosed hereinwill be similarly useful as mycoattractants and/or mycopesticides withsuch beetles. By way of example but not of limitation, such beetlesinclude bark, sap and wood-boring beetles such as the mountain pinebeetle (Dendroctonus ponderosae), spruce beetle (Dendroctonusrufipennis), red turpentine beetle (Dendroctonus valens), blackturpentine beetle (Dendroctonus terebrans), southern pine beetle(Dendroctonus frontalis), Douglas fir beetle (Dendroctonuspseudotsugae), engraver and Ips beetles including Ips avulsus, Ipsgrandicollis, Ips calligraphus, Ips pini, Ips avulses, and other sapbeetles in the family Nitidulidae, powderpost beetles (Lyctidae), falsepowderpost beetles (Bostrichidae), deathwatch beetles, oldhouse borers,Asian long-horned beetle, etc.

It is further expected that the preconidial products and methods may,with no more than routine experimentation, prove useful againstpresocial, parasocial and subsocial insects including semisocial,quasisocial, communal and solitary insect pests such as cockroachesincluding American, German, Surinam, brown-banded, smokybrown, and Asiancockroaches, grasshoppers and locusts, crickets including mole cricket,Mormon crickets (actually a long-horned grasshopper), beetles, beetlegrubs and beetle larvae including Colorado potato beetle (Leptinotarsadecemlineata) and other potato beetles, Mexican bean beetle, Japanesebeetle, cereal leaf beetle, darkling beetle (lesser mealworm), Gypsymoths (Lymantria dispar) and Gypsy moth larvae, diamondback moths(Plutella xylostella), codling moth (Laspeyresia pomonella), Douglas firtussock moth (Orgyia pseudotsugata), western spruce budworm(Choristoneura occidentalis), grape berry moths (Lobesia lobina), fliesand fly larvae, springtails, large centipedes, shield centipedes,millipedes, European corn borers (Ostrinia nubilalis), Asiatic cornborers, velvetbean caterpillar (Anticarsia gemmatalis), othercaterpillars and larvae of the Lepidoptera, whiteflies (Dialeurodes andBemisia spp.), thrips (Thrips spp.), melon thrips (Thrips palmi),western flower thrips (Frankliniella occidentalis), aphids includingRussian wheat aphid, spider mites (Tetranychus spp.), mealybugsincluding citrus mealybug (Planococcus citri) and solanum mealybug(Pseudococcus solani), boll weevils, black vine weevils (Otiorhynchussulcatus), European pecan weevils (Curculio caryae), mosquitoes, wasps,sweet potato whiteflies, silverleaf whiteflies, cotton fleahoppers,pasture scarabs such as Adoryphorus couloni and other Scarabaeidae,spittle bug (Mahanarva posticata), corn earworm (Helicoverpa zea),American bollworm (Heliothis armigera), armyworms (Pseudaletiaunipuncta), fall armyworm (Spodoptera frugiperda), southern armyworm(Spodoptera eridania), beet armyworm (Spodoptera exigua), yellowstripedarmyworm (Spodoptera omithogalli), black cutworm (Agrotis ipsilon),tobacco hornworm (Manduco Sexta), tobacco budworm (Helicoverpa (syn.Helicoverpa) virescens), sugar cane froghopper, rice brown planthopper,earwigs, loopers including cabbage looper (Trichoplusia ni), soybeanlooper (Pseudoplusia includens), forage looper (Caenurgina erechtea) andcelery looper (Anagraph afalcifera), cabbageworms including the importedcabbageworm (Pieris rapae) and the European cabbageworm (Pieriesbrassicae), tomato pinworm (Keiferia lycopersicella), tomato hornworm(Manduca quinquemaculata), leafminers (Liriomyza spp.), cotton leafworm(Alabama argillacea), corn rootworm, garden webworm (Achyra rantalis),grape leaffolder (Desmia funeralis), melonworm (Diaphania hyalinata),pickleworm (Diaphania nitidalis), achemon sphinx (Eumorpha achemon),sweetpotato hornworm (Agrius cingulata), whitelined sphinx (Hyleslineata), lygus bugs (Lygus spp.), chinch bugs including Blissusleucopterus and false chinch bugs, sow bugs, pill bugs, citrus rustmite, pill wood lice, wheat cockchafer, white grubs and cockchafers,Hoplochelis marginalis and Melolontha melontha, storage pests such asProstephanus truncatus and Sitophilus zeamais, soil insects, and variousother insect pests in the orders, Isopoda, Diplopoda, Chilopoda,Symphyla, Thysanura, Collembola, Orthoptera, Dermaptera, Anoplura,Mallophaga, Thysanoptera, Heteroptera, Homoptera, Lepidoptera,Coleoptera, Diptera, Siphonaptera, Thysaoptera, Acarina, Arachnida, etc.and the families Plutellidae, Acrididae, Tettigoniidae, Gryllidae,Cryllotalpidae, Pyralidae, Sphingidae, Noctuidae, Pyralidae,Xylophagidae, Scarabaeidae, Scolytidae, Platypodidae, Lymexylidae,Nitidulidae, Pseudococcidae, Aphidae, Dalphacidae, Cicadellidae,Cercopidae, Aleyodidae, Coccoidea, etc. It will be recognized that theinsects listed above are representative examples of insects which may beattracted and/or controlled according to the present invention, but suchlisting is not intended as a limitation to certain species as numerousother insect species to which the invention may be applied will beapparent to those skilled in the art.

It will be noted from the discussion above and examples and resultsbelow that attractiveness, pathogenicity and virulency toward thetargeted insect are dependent in some degree upon factors includingchoice of mycopesticidal species, host range and specificity, selectionof a strain within that species and selection of substrate.Entomopathogenic fungi also vary greatly in host specificity. Someentomopathogenic fungi are highly specific, such as Pandora neoaphidis,which is restricted to aphids. Other entomopathogenic fungi have widehost ranges, such as Beauveria bassiana, which is known to infect over700 species of arthropods. Other species with wide host ranges includeMetarhizium anisopliae, Paecilomyces farinosus and Zoophthora radicans.However, in the laboratory, isolates of fungi with wide host ranges aregenerally most virulent to the host from which they were first isolated;certainly their host range is much more restricted than that of thespecies to which they belong. Goettel et al., Safety to NontargetInvertebrates of Fungal Biocontrol Agents, in: Laird et. al. (eds.)Safety of Microbial Insecticides, pp 209-232 (1990). Furthermore, fungiwith wide host ranges are frequently even more specific under fieldconditions. There are reports of fungi attacking only one host eventhough closely related host species are present. Discrepancies betweenreports of social insect host specificity may be related to a generaldifference between tropical vs. temperate habitats rather than to thespecific fungi and social insect species involved. Schmid-Hempel, supraat p. 44. Such specificity is thought to be due to the complex bioticand abiotic interactions in the field. This indicates that it should bepossible, using no more than routine experimentation and bioassays ofmycopesticidal strains and of the appropriate orders, families, genera,species and varieties of targeted pest insects, to isolate and usestrains and substrates wherein the desired characteristics are maximizedwith respect to either a targeted insect or targeted insect group,thereby producing a species-specific, genus-specific, family-specific ororder-specific entomopathogenic host specific fungal strain. Suchentomopathogenic strains selected for host range and specificity may besimilarly selected for minimal or no infection of or virulence towardsbeneficial insects or non-targeted insects.

For initial experimentation, a Metarhizium anisopliae from naturallyoccurring sources and the carpenter ant were selected. M. anisopliae wasobtained from a public culture collection and used without furtherselection for virulence and/or pathogenicity; a publicly availablestrain free of proprietary or patent restrictions on use was selected asoffering a preferred source and a more demanding initial test thanstrains selected for specific virulence. It will be understood, ofcourse, that strains selected for specific characteristics ofattractiveness to and/or virulence against specific insects will ingeneral offer the best mode of practicing the invention. Cultures may beobtained from collections, isolated from the wild and/or reisolated frominsects. The carpenter ant was selected for initial experimentation asoffering several advantages: ants are typically more resistant to sporesthan termites and other insects, carpenter ants are a very destructivepest, the effect on other ant species could also be viewed, and theapplicant enjoyed easy access to an experimental site as his residencewas in danger of collapse due to long term structural infestation bycarpenter ants and fungi.

Example 1

Metarhizium anisopliae ATCC #62176 was grown in pure culture usingstandard fermentation techniques and diluted and aseptically transferredto grain (brown rice) which had been pressure steam-sterilized at one(1) kg/cm² (15 psi). The mycelium overgrew the rice and approximately10-20 grams of preconidial mycelium of Metarhizium anisopliae wasoffered at the site of debris piles caused by carpenter ants (Camponotusmodoc) at the interior face of an exterior wall of a wood frameresidence located in Shelton, Wash., U.S.A. The mycelium was presentedon a small dish and left exposed to the air. An observation made afterthree hours disclosed the carpenter ants feasting en masse on thenon-sporulating, preconidial mycelium of the Metarhizium andapproximately one dozen (12) carpenter ants were observed retreatinginto the wall, carrying pieces of the infectious mycelium with them. Ina week's time, the carpenter ant colony became inactive and no evidenceof carpenter ant activity was observed henceforth, saving the structurefrom further structural damage. Months later, the ecological niche onceoccupied by the carpenter ants was taken over by common household Sugarand Honey ants which were unaffected by the Metarhizium anisopliae.Carpenter ants have not been observed in the residence in the subsequenttwo years even though they are plentiful in a woodpile outside thehouse.

Example 2

For “choice” tests, termite colony fragments of 50 pseudergates(workers) of the Eastern Subterranean Termite Reticulitermes flavipes(Kollar) or Formosan Subterranean Termite Coptotermes formosanus(Shiraki) [Isoptera: Rhinotermitidae] per test unit arena were collectedprior to the start of the bioassay evaluation. The termite colonyfragments were placed in plastic boxes with soil, adjusted to laboratoryconditions and fed standard diet (standard tongue depressor section) andprovided with a moistened cellulose source placed on top of the fungalpreconidial mycelium in hexagonal weigh boats, perforated with 5 mmholes on all sides to allow termite entry. For “no-choice” or tubetests, termite colony fragments of 50 pseudergates (workers) per tubewere collected prior to the start of the bioassay evaluation. Glasstubes were prepared containing fungal preconidial mycelium in thecenter, with moistened soil on each end of the mycelium, then bounded oneach end by agar plugs. The bottom of the tube contained a 3 cm sectionof applicator stick, and was capped with foil and rubber banded. Termitecolony fragments of 50 pseudergates were placed in the top section,above the agar plug, the end was capped with foil and rubber banded andobservations were made as they tunneled down through the agar plug, toplayer of soil, mycelium/rice mixture, and bottom layer of soil.Treatment was with preconidial mycelium products consisting ofMetarhizium anisopliae ATCC #62176 on rice, Beauveria bassiana ATCC #20872 on rice and Beauveria bassiana ATCC #74038 on rice. There werefour replicates of each of the test arenas and tubes for each termitespecies, and four replicates of control arenas, for a total of 16arenas. Mortality (%) observations were recorded for both arena and tubebioassays.

Metarhizium anisopliae #62176 on rice was tunneled freely by bothReticulitermes and Coptotermes spp. in the test arenas. No significantmortality for either species was observed in the control or treatmentarenas through day 28 of the evaluation.

Beauveria bassiana #20872 on rice was tunneled freely by bothReticulitermes and Coptotermes spp. with significant mortality in bothReticulitermes and Coptotermes. In the arenas, both species of termitesbuilt shelter tubes or soil connectives to the wood in the controlsamples and to the preconidial mycelium/rice product in the treatmentsamples. In the arena bioassay, significant mortality was observed inReticulitermes, with a mean value of 52.5% for Beauveria #20872 on ricevs. a mean value of 1.25% for controls. In the arena bioassay withCoptotermes, significant mortality was observed for Beauveria #20872 onrice, with a mean value of 100% for treatments vs. a mean value of 2.5%for the controls.

In the tube bioassay, significant mortality was observed inReticulitermes, with a mean mortality value of 65.5% for treatment withBeauveria #20872 on rice vs. a mean value of 1.67% for the controls. InCoptotermes, with Beauveria #20872 on rice, a mean value of 100% for thetreatments vs. a mean value of 9.17% for the controls was obtained. Bothspecies of termites tunneled through the control soil samples and agarsections. In the treatment samples, both species of termites tunneledthrough the top agar section and upper soil layer into the mycelium/riceproduct treatment area. Reticulitermes flavipes continued through thetreatment and lower soil and agar section and placed soil around thewooden stick at the bottom, which is typical behavior of subterraneantermites in a tube bioassay. Coptotermes formosanus termites, on theother hand, never left the mycelium/rice product treatment area.Atypically, they did not continue to tunnel through the lower soil andagar section to reach the wooden stick at the bottom of the tube. Theywere observed to remain in the preconidial mycelium test product, and atthe end of the two week period, mortality levels were recorded at 100%.

With Reticulitermes utilizing Beauveria bassiana #20872 preconidialmycelium on wooden blocks, termites foraged to the blocks immediately.Within five days mortality began to be observed in the arenas withpreconidial mycelium and mean mortalities of 90% and 100% were observedover 14 days.

Example 3

Test unit colony fragments of 30 pseudergate Reticulitermes flavipes(Kollar) Eastern subterranean termites per tube bioassay wereestablished as above and broken down after 14 days testing. There werethree replicates of each of three test preconidial mycelium test strainsand three control tubes, for a total of 12 tubes.

Beauveria bassiana #74038 on rice was freely tunneled in test arenas bythe termites. Significant mortality (100%) was observed. A mortality of45.56% was observed in controls.

Beauveria bassiana #20872 on rice was freely tunneled in test arenas bythe termites. Non-significant mortality levels of 84.4% were observedwith control group mortality of 45.56%.

Metarhizium anisopliae #62176 on rice was freely tunneled in test arenasby the termites. Non-significant mortality levels of 66.3% were observedwith control group mortality of 45.56%.

Eastern Subterranean Termite % Mortality at 14 Days (3 replicates)Strain # 74038 100 100 100 Strain # 20872 53.3 100 100 Strain # 6217676.67 30.00 92.34 Control 56.67 23.34 56.67

Example 4

Colony fragments of 100 Solenopsis invicta (Buren) red imported fireants per standard plastic box test arena were established with moistenedsoil and preconidial mycelium. Three drops of water were added to thesoil daily to maintain a humid medium amenable to ant activity and fugalgrowth. Beauveria bassiana #20872 and #74038 and Metarhizium anisopliae#62176 preconidial mycelium on rice were utilized as test strains. Therewere three replicates of each of three test strains and three controlarenas, for a total of 12 arenas. In all test arenas portions of thepreconidial mycelium were removed readily from the feeding dishes by theants and scattered over the arena floors. Ants also readily moved intoand created gallery-like tunnels in the preconidial mycelium on rice inthe feeding dishes.

When considering the mortality numbers for all three strains, there wasnot a statistically significant difference from the control group at 7days or 14 days post-association. This was also true at 21 days for#20872 and #62176.

% Mortality (Mean value of 3 replicates) Red Imported Fire Ants 7 Days14 Days 21 Days Strain # 39.33 80.33 98.00 74038 Strain # 35.66 76.6688.66 20872 Strain # 39.66 62.66 82.33 62176 Control 31.00 44.66 66.33 %Mortality (3 replicates) Red Imported Fire Ants 7 Days 14 Days 21 DaysStrain # 38 76 98 74038 32 80 100 48 85 96 Strain # 39 83 94 20872 42 84100 26 63 72 Strain # 29 38 55 62176 57 94 96 33 56 96 Control 54 71 8214 24 46 25 39 71

Example 5

Cultivate strains of Metarhizium, Beauveria and Cordyceps on grain,wood, or other cellulosic substrate as above under elevated CO₂conditions to produce preconidial mycelium. Apply as attractant and/orpathogen at locations infested by insects such as carpenter ants,termites, beetles, flies, fire ants, cockroaches, grasshoppers, locustsand other insect pests and vermin.

Example 6

In separate glass containers, extracts of Beauveria bassiana strainsATCC #20872 and ATCC #74038 were created from fresh preconidial myceliagrown out on organic short grain brown rice in spawn bags incubated for11 days in class 100 clean room using the following procedures.

2,140 gm. (4.70 lb.) of ATCC #20872 Beauveria bassiana mycelium on ricewas combined with 2,100 ml. (3.55 lb.) 95% (190 proof) organic grainalcohol diluted with 1,100 ml. (2.25 lb.) of spring water and maceratedand incubated for a total of 14 days. Alcohol measured 45% at 14 days.An unusual characteristic, fragrance signature reminiscent of vanillaand Pepsi® cola was noted.

2,120 gm. (4.65 lb.) of ATCC #74038 Beauveria bassiana mycelium on ricewas combined with 2,050 ml. (3.40 lb.) 95% (190 proof) organic gainalcohol diluted with 1,025 ml. (2.10 lb.) of spring water and maceratedand incubated for a total of 14 days. Alcohol measured 50% at 14 days. Acharacteristic fragrance signature of old-fashioned hard Christmas candywas noted.

Example 7

Alcohol extract of ATCC #74038 from Example 6 above was applied to woodwhich was placed in a test arena with Formosan termites, Coptotermesformosanus. Termites were observed streaming toward the bait. Theycontinued to struggle to reach the bait as they were overcome by thealcohol fumes prior to death.

Example 8

1) Extract fresh preconidial mycelium of an entomopathogenic fungi, forexample Beauveria, Metarhizium or Cordyceps grown on grain, wood orpaper, with 100% (anhydrous) or 95% (water/alcohol azeotrope) ethanolutilizing a Soxhlet or other extraction apparatus. Optionally remove theethanol with a rotary evaporator or other apparatus prior to applicationand use.

2) Extract fresh entomopathogenic preconidial mycelium with a non-polarsolvent, for example dichloromethane or toluene. Then extract thepreconidial mycelium with a less polar solvent, for example acetone orisopropyl alcohol. Remove solvents under vacuum to obtain extracts.

3) Extract fresh preconidial mycelium on substrate with an organicsolvent or mixture of solvents to obtain extracts. Effect furtherpurification if desired through chromatography, vacuum distillationand/or other purification methods. Isolate and characterize attractantfractions and compounds if desired. Synthesize attractant molecules,isomers and analogs if desired.

4) Place fresh preconidial entomopathogenic mycelium on grain in a flaskand steam distill utilizing boiling water or introduced steam orsuperheated steam. Collect the condensate and separate steam-volatileattractant components from the aqueous phase utilizing separatory funnelor other apparatus. Extract aqueous phase with water-immiscible organicsolvent or utilize “attractant water” as is.

No limitations with respect to the specific embodiments and examplesdisclosed herein are intended or should be inferred, as the examples andembodiments are representative only. While examples and preferredembodiments of the present invention have been shown and described, itwill be apparent to those skilled in the art, or ascertainable using nomore than routine experimentation, that many changes and modificationsmay be made without departing from the invention in its broader aspects.The appended claims are therefore intended to cover all such changes,modifications and equivalents as fall within the true spirit and scopeof the invention.

1. An entomopathogenic fungal composition comprising an extract of apreconidial mycelium of an entomopathogenic fungus cultivated on a solidsubstrate wherein the preconidial mycelium is cultured from a sector ofan entomopathogenic fungus culture displaying the preconidial myceliumrather than those sectors displaying post-conidial mycelium and whereinthe entomopathogenic fungus is selected from the group consisting ofMetarhizium, Beauveria, Paecilomyces, Hirsutella, Verticillium,Culicinomyces, Nomuraea, Aspergillus, Cordyceps, Ascosphaera,Torrubiella, Hypocrella and its Aschersonia anamorph, Entomophaga,Massospora, Neozygites, Zoophthora, Pandora and Laccaria species andcombinations thereof.
 2. The entomopathogenic fungal composition ofclaim 1 wherein the entomopathogenic fungus is selected from the groupconsisting of Metarhizium anisopliae, Metarhizium flaviride, Beauveriabassiana, Beauveria brongniartii, Paecilomyces farinosus, Paecilomycesfumosoroseus, Verticillium lecanii, Hirsutella citriformis, Hirsutellathompsoni, Aschersonia aleyrodis, Entomophaga grylli, Entomophagamaimaiga, Entomophaga muscae, Entomophaga praxibulli, Entomophthoraplutellae, Zoophthora radicans, Neozygites floridana, Nomuraea rileyi,Pandora neoaphidis, Tolypocladium cylindrosporum, Culicinomycesclavosporus, Lagenidium giganteum, Cordyceps variabilis, Cordycepsfacis, Cordyceps subsessilis, Cordyceps myrmecophila, Cordycepssphecocephala, Cordyceps entomorrhiza, Cordyceps gracilis, Cordycepsmilitaris, Cordyceps washingtonensis, Cordyceps melolanthae, Cordycepsravenelii, Cordyceps unilateralis, Cordyceps sinensis, Cordycepsclavulata, Laccaria bicolor and combinations thereof.
 3. Theentomopathogenic fungal composition of claim 1 wherein the solidsubstrate is selected from the group consisting of grains, seeds, wood,paper products, cardboard, sawdust, corn cobs, cornstalks, chip board,hemp, jute, flax, sisal, reeds, grasses, bamboo, papyrus, coconutfibers, nut casings, seed hulls, straws, sugar cane bagasse, soybeanroughage, coffee wastes, tea wastes, cactus wastes, banana fronds, palmleaves, fiberized rag stock, fabrics, landscaping cloths, geofabrics,soil blankets and rugs, mats, mattings, bags, baskets, gabions, fiberlogs, fiber bricks, fiber ropes, nettings, felts, tatamis andcombinations thereof.
 4. The insect attractant and phagostimulantcomposition of claim 1 wherein the solid substrate is selected from thegroup consisting of cellulosic substrates, ligninic substrates,celluloligninic substrates, carbohydrate substrates and combinationsthereof.
 5. The entomopathogenic fungal composition of claim 1 whereinthe preconidial mycelium is metabolically arrested via a method selectedfrom the group consisting of drying, freeze-drying, refrigerating,gaseous cooling, light deprivation, cryogenic suspension andcombinations thereof.
 6. The entomopathogenic fungal composition ofclaim 1 wherein the composition further comprises an insect controlagent selected from the group consisting of biological control agents,chemical control agents, physical control agents and combinationsthereof.
 7. The entomopathogenic fungal composition of claim 1 whereinthe extract is extracted with a solvent selected from the groupconsisting of water, steam, alcohols, organic solvents, carbon dioxideand combinations thereof.
 8. The entomopathogenic fungal composition ofclaim 7 wherein the organic solvents are selected from the groupconsisting of alcohols containing from 1 to 10 carbon atoms,unsubstituted organic solvents containing from 1 to 16 carbon atoms,ketones containing from 3 to 13 carbon atoms, ethers containing from 2to 15 carbon atoms, esters containing from 2 to 18 carbon atoms,nitriles containing from 2 to 12 carbon atoms, amides containing from 1to 15 carbon atoms, amines and nitrogen-containing heterocyclescontaining from 1 to 10 carbon atoms, halogen substituted organicsolvents containing from 1 to 14 carbon atoms, acids containing from 1to 10 carbon atoms, and alkoxy, aryloxy, cyloalkyl, aryl, alkaryl andaralkyl substituted organic solvents containing from 3 to 13 carbonatoms and combinations thereof.
 9. The entomopathogenic fungalcomposition of claim 7 wherein the organic solvents are selected fromthe group consisting of methanol, ethanol, isopropanol, n-propanol,n-butanol, 2-butanol, 2-methyl-1-propanol, ethylene glycol, glycerol,benzene, cyclohexane, cyclopentane, methylcyclohexane, pentanes,hexanes, heptanes, 2,2,4-trimethylpentane, toluene, xylenes, acetone,2-butanone, 3-pentanone, 4-methyl-2-pentanone, t-butyl methyl ether,1,4-dioxane, diethyl ether, tetrahydrofuran, methyl formate, ethylacetate and butyl acetate, acetonitrile, proprionitrile, benzonitrile,formamide, N,N-dimethylformamide, N,N-dimethylacetamide, pyrrolidine,1-methyl-2-pyrrolidinone, pyridine, bromotrichloromethane, carbontetrachloride, chlorobenzene, chloroform, 1,2-dichloroethane,dichloromethane, 1-chlorobutane, trichloroethylene, tetrachloroethylene,1,2-dichlorobenzene, 1,2,4-trichlorobenzene,1,1,2-trichlorotrifluoroethane, 2-butoxyethanol, 2-ethoxyethanol,ethylene glycol dimethyl ether, 2-methoxyethanol, 2-methoxyethyl ether,2-ethoxyethyl ether, acetic acid, trifluoroacetic acid, carbondisulfide, methyl sulfoxide, nitromethane and combinations thereof. 10.The entomopathogenic fungal composition of claim 1 wherein the extractis prepared with a polar solvent.
 11. An entomopathogenic fungalcomposition comprising an extract of an entomopathogenic fungus myceliumcultured on a solid substrate, wherein the entomopathogenic fungusmycelium is in a developmental state prior to spore formation, whereinthe entomopathogenic fungus mycelium is cultured from a sector of anentomopathogenic fungus culture displaying pre-sporulation mycelium, andwherein the entomopathogenic fungus is selected from the groupconsisting of Metarhizium, Beauveria, Paecilomyces, Hirsutella,Verticillium, Culicinomyces, Nomuraea, Aspergillus, Cordyceps,Ascosphaera, Torrubiella, Hypocrella and its Aschersonia anamorph,Entomophaga, Massospora, Neozygites, Zoophthora, Pandora and Laccariaspecies and combinations thereof.